Influence of cyclin type and dose on mitotic entry and progression in the early Drosophila embryo
Cyclins are key cell cycle regulators, yet few analyses test their role in timing the events that they regulate. We used RNA interference and real-time visualization in embryos to define the events regulated by each of the three mitotic cyclins of Drosophila melanogaster, CycA, CycB, and CycB3. Each individual and pairwise knockdown results in distinct mitotic phenotypes. For example, mitosis without metaphase occurs upon knockdown of CycA and CycB. To separate the role of cyclin levels from the influences of cyclin type, we knocked down two cyclins and reduced the gene dose of the one remaining cyclin. This reduction did not prolong interphase but instead interrupted mitotic progression. Mitotic prophase chromosomes formed, centrosomes divided, and nuclei exited mitosis without executing later events. This prompt but curtailed mitosis shows that accumulation of cyclin function does not directly time mitotic entry in these early embryonic cycles and that cyclin function can be sufficient for some mitotic events although inadequate for others.
THE JOURNAL OF CELL BIOLOGY
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J. Cell Biol. Vol. 184 No. 5 639–646
Correspondence to Patrick H. O ’ Farrell: firstname.lastname@example.org
Abbreviations used in this paper: dsRNA, double-stranded RNA; TACC, trans-
forming acidic coiled coil.
Since the discovery of the cell cycle – coupled accumulation and
destruction of cyclins ( Evans et al., 1983 ), the increase in these
mitotic regulators has been discussed as a possible clock timing
cell cycle progression ( Murray and Kirschner, 1989 , 1991 ).
Although other regulatory inputs (notably relief of inhibitory phos-
phorylation of the mitotic cyclin partner Cdk1) have been
recognized as triggers of mitotic entry ( Russell and Nurse, 1986 ;
Edgar and O ’ Farrell, 1990 ; O ’ Farrell, 2001 ), the realization that
the mechanisms controlling progress of the cell cycle change dur-
ing development opened the question of what controls mitotic
entry at other stages ( O ’ Farrell et al., 1989 ). In particular, early
embryonic cell cycles, which typically are exceedingly fast and
run independent of new gene expression, occur with little or no
Cdk1 inhibitory phosphorylation in frogs and ies ( Ferrell et al.,
1991 ; Edgar et al., 1994 ). These cycles, which also lack gap
phases, have been viewed as streamlined cycles that operate with-
out many of the controls that limit cell cycle progress later in de-
velopment, and it has been asserted that these cycles are driven by
a cyclin oscillator ( Murray and Kirschner, 1991 ; Murray, 2004 ).
However, there is very little in vivo experimental support for the
in uential view that interphase duration of embryonic cycles is
determined by the time required to accumulate cyclin to a thresh-
old ( Hartley et al., 1996 ).
Developmental changes in cell cycle regulation have been
detailed in Drosophila melanogaster . The cell cycle acquires a
G2 and slows at embryonic cycle 14 as a result of increased
Cdk1 inhibitory phosphorylation ( Edgar and O ’ Farrell, 1989 ).
Programmed transcription of the activating phosphatase String
(Cdc25) times entry into mitosis from this G2 ( Edgar and
O ’ Farrell, 1989 , 1990 ). However, string is abundant throughout
the earlier mitotic cycles, which occur in a syncytial cytoplasm
without cytokinesis and feature rapid oscillation between DNA
replication and mitosis. In accord with the consensus view, it
has been suggested that cyclin accumulation times these cycles
( O ’ Farrell et al., 1989 ). Indeed, it has been shown that the dose
of maternal genes encoding cyclins in uences the speed of the
early mitotic cycles ( Edgar et al., 1994 ; Stif er et al., 1999 ;
Ji et al., 2004 ; Crest et al., 2007 ) and that mitotic exit requires
cyclin destruction ( Su et al., 1998 ). However, the bulk levels of
mitotic cyclin exhibit little oscillation during the earliest embry-
onic cycles ( Edgar et al., 1994 ), and the in uence of cyclin dose
on interphase length does not follow the direct proportional-
ity predicted by the cyclin oscillator model. The combination
of maternal contribution of cyclins to the egg and germline
requirements for cyclin has prevented more complete genetic
analyses of cyclin function during the early divisions, but analyses
yclins are key cell cycle regulators, yet few analy-
ses test their role in timing the events that they
regulate. We used RNA interference and real-
time visualization in embryos to deﬁ ne the events regu-
lated by each of the three mitotic cyclins of Drosophila
melanogaster , CycA, CycB, and CycB3. Each individual
and pairwise knockdown results in distinct mitotic pheno-
types. For example, mitosis without metaphase occurs
upon knockdown of CycA and CycB. To separate the
role of cyclin levels from the inﬂ uences of cyclin type, we
knocked down two cyclins and reduced the gene dose of
the one remaining cyclin. This reduction did not prolong
interphase but instead interrupted mitotic progression.
Mitotic prophase chromosomes formed, centrosomes di-
vided, and nuclei exited mitosis without executing later
events. This prompt but curtailed mitosis shows that ac-
cumulation of cyclin function does not directly time mi-
totic entry in these early embryonic cycles and that cyclin
function can be sufﬁ cient for some mitotic events al-
though inadequate for others.
Inﬂ uence of cyclin type and dose on mitotic entry
and progression in the early Drosophila embryo
Mark L. McCleland , Jeffrey A. Farrell , and Patrick H. O ’ Farrell
Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143
© 2009 McCleland et al. This article is distributed under the terms of an Attribution–
Noncommercial–Share Alike–No Mirror Sites license for the ﬁ rst six months after the publica-
tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a
Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license,
as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
JCB • VOLUME 184 • NUMBER 5 • 2009 640
Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/
jcb.200810012/DC1). GFP-Polo is centrosomal in interphase, but
at the rst sign of mitosis, it enters the nucleus where it decorates
kinetochores (Video 2). During chromosome alignment, kineto-
chore staining declines, and upon anaphase, GFP-Polo accumu-
lates at the spindle midzone ( Moutinho-Santos et al., 1999 ).
After simultaneous knockdown of CycA and CycB, leav-
ing CycB3, mitosis occurred in the absence of metaphase.
Chromatin condensed and persisted for ⵑ 3 min in a central
spherical mass without forming a characteristic rectangular
cluster of metaphase chromosomes, then two chromosomal
masses segregated to opposite poles, although the distribution
was often unequal, and lagging chromosomes were frequent
( Fig. 1 B; Fig. 2 A ; and Video 3, available at http://www.jcb
.org/cgi/content/full/jcb.200810012/DC1). During this CycB3-
directed mitosis, GFP-Polo entered the nucleus during prophase
and localized to the spindle midzone upon mitotic exit ( Fig. 1 B
and Video 4). Kinetochores accumulated much less GFP-Polo,
and they failed to achieve a metaphase alignment. Thus, CycA
and CycB normally promote metaphase, and CycB3 lacks this
capability under these conditions.
Simultaneous knockdown of CycB and CycB3, leaving
CycA, doubled the duration of chromosome alignment and
metaphase ( Fig. 1; Fig. 2 A ; and Video 5, available at http://
chromosome separation was slow, truncated, and accompanied
by decondensation. Nuclei typically snapped back together in
telophase and subsequently fell to the embryo interior. GFP-
Polo displayed robust prometaphase kinetochore localization
and faded only slowly during the extended metaphase ( Fig. 1 C
and Video 6). Often, anaphase kinetochores appeared to remain
attached to spindle remnants rather than to centrosomes ( Fig. 1 C ,
arrows). The prolongation of early mitotic events, slowed chro-
mosome separation, and truncated mitotic exit suggest that
CycB and/or CycB3 normally promote the transition to and
execution of anaphase.
Embryos injected with CycA and CycB3 dsRNA, leaving
CycB, exhibited a more mild disruption of mitotic coordination.
Chromosome alignment was slightly delayed and metaphase
duration doubled, but nonetheless, nuclei successfully com-
pleted mitosis ( Fig. 2 A ). Thus, CycB can mediate the key
events of mitosis. Knockdown of just CycB interfered with chro-
mosome congression and resulted in anaphase failures (Fig. S1
and Videos 7 and 8, available at http://www.jcb.org/cgi/content/
full/jcb.200810012/DC1). The resulting nuclei ultimately fell to
the embryo interior. Thus, CycB is essential for the syncytial
Advances in confocal microscopy, RNAi methodology,
and GFP biomarkers have allowed us to capture temporal re-
cords of the altered mitoses resulting from cyclin reduction.
In this way, our data go beyond the previous analysis of cyclin
mutants using xed embryos. Furthermore, we have charac-
terized cyclin function during the early rapid mitotic cycles,
whereas previous work focused on cyclin roles during later cy-
cles ( Knoblich and Lehner, 1993 ; Jacobs et al., 1998 ) in which
mitotic entry is governed by String expression. Perhaps because
of differences in methodology or stage, two of our observations
of mutations revealed cyclin roles during later zygotically con-
The three mitotic cyclins of Drosophila are degraded in
succession, CycA before metaphase, CycB at the metaphase –
anaphase transition, and CycB3 during anaphase ( Sigrist et al.,
1995 ). Mutants lacking CycB or CycB3 produce viable ies
( Lehner and O ’ Farrell, 1990 ; Jacobs et al., 1998 ). CycA is re-
quired for mitosis 16 because of an unexpected interphase role
rather than a mitotic requirement ( Lehner and O ’ Farrell, 1989 ;
Sigrist et al., 1995 ; Reber et al., 2006 ). Despite dispensability of
the individual cyclins, the mitotic phenotypes of cyclin double
mutants in the postsyncytial divisions (cycles 15 and 16) indi-
cate that individual requirements are partially covered by redun-
dancy ( Lehner and O ’ Farrell, 1990 ; Knoblich and Lehner, 1993 ;
Jacobs et al., 1998 ). The mutant phenotypes and the differing
consequences of stabilizing each of the cyclins revealed special-
izations ( Sigrist et al., 1995 ; Jacobs et al., 1998 ; Parry and
O ’ Farrell, 2001 ). It was proposed that, rather than acting as ge-
neric promoters of mitosis, the specialized actions of the mitotic
cyclins are modulated by their schedules of destruction to guide
progression through mitosis. However, this model ascribing
speci c roles to the cyclins is based mostly on xed analyses,
which have left us with only a super cial understanding of each
cyclin ’ s function.
In this report, we show the specialized role of each mitotic
cyclin based on real-time analysis after cyclin RNAi in the early
syncytial embryo. Knockdown of any two cyclins modestly ex-
tended interphase but did not prevent mitotic entry. Surpris-
ingly, lowering cyclin function even further by halving the
maternal dose of the remaining cyclin did not further extend
interphase as would be predicted by the cyclin accumulation
model. Instead, nuclei entered a curtailed mitosis, wherein
chromosomes temporarily condensed, and centrosomes divided
without nuclear envelope breakdown or nuclear division. Ap-
parently, nuclei attempted mitosis while still lacking cyclin
function adequate for proper mitotic execution. These data sug-
gest that cyclin accumulation to a speci c threshold does not
directly time Cdk1 activation.
Results and discussion
Cyclin contributions to mitotic progression
RNAi in Drosophila embryos ( Yang et al., 2000 ; Echard and
O ’ Farrell, 2003 ) results in particularly rapid, speci c, and effec-
tive knockdown of mitotic cyclins during the blastoderm divi-
sions ( McCleland and O ’ Farrell, 2008 ). Embryos (cycles 8 – 10)
were injected at one pole with single and all pairwise combina-
tions of cyclin double-stranded RNA (dsRNA) and imaged in
real time through interphase 14. Unlike the triple-cyclin knock-
down ( McCleland and O ’ Farrell, 2008 ), none of these knock-
downs prevented mitosis, although it was delayed and its
progression was altered ( Fig. 1 and Fig. 2 A ). Gradations in the
severity of phenotypes occurred in accord with proximity to the
dsRNA injection site and with time after injection (see Materials
Histone-GFP marks mitotic chromosome behavior, whereas
dynamic changes in GFP-Polo mark several events ( Fig. 1 and
641CELL CYCLE EFFECTS AFTER CYCLIN REDUCTION • McCleland et al.
cyclin has generally been interpreted to mean that anaphase onset
is independent of cyclin degradation and requires only separase
activation ( Holloway et al., 1993 ); however, the particular capa-
bility of the tested cyclin to support different mitotic stages will
in uence the result. We nd that CycB3 cannot support meta-
phase. Furthermore, reduction of CycB3 alone or in combination
with other cyclins extended metaphase, suggesting that CycB3
promotes the transition to anaphase ( Fig. 2 A ). These ndings
support an interpretation of the stable cyclin results that empha-
sizes the distinctions in their activities and suggests that the
normal successive destruction of the different cyclins modulates
cyclin/Cdk1 activity so as to promote the normal sequence of
mitotic events ( Fig. 2 B ; Jacobs et al., 1998 ).
differ from previous ndings. First, we show that CycB is
essential for the success of syncytial mitosis but that it is zy-
gotically dispensable. Second, we observed the unusual meta-
phaseless mitosis upon knockdown of CycA and CycB, whereas
the double mutant of CycA and CycB arrested in interphase
( Knoblich and Lehner, 1993 ; Jacobs et al., 1998 ).
It is also interesting to compare our ndings to the gain of
function phenotypes observed upon expression of individual sta-
bilized cyclins ( Sigrist et al., 1995 ; Parry and O ’ Farrell, 2001 ).
Each stabilized cyclin arrested progress of mitosis at different
points, stable CycA in metaphase, and stabilized CycB or CycB3
in distinct stages of anaphase ( Parry and O ’ Farrell, 2001 ; Parry
et al., 2003 ). Progress to anaphase in the presence of a stabilized
Figure 1. Mitotic cyclins provide both redundant and speciﬁ c function during mitosis. (A – C) Embryos expressing histone-GFP (top) or GFP-Polo (bottom)
were imaged through mitosis 13. (A) Control embryos injected with LacI dsRNA. (B) Embryos injected with CycA and CycB dsRNA. Note that chromosomes
fail to congress (02:36) or properly recruit GFP-Polo onto kinetochores (02:23 and 04:23). (C) Embryos injected with CycB and CycB3 dsRNA. Note
that nuclei exhibit a prolonged metaphase (06:26), extended GFP-Polo concentration on kinetochores in metaphase (08:42), and a truncated anaphase.
Arrows highlight kinetochores that fail to reach centrosomes. The times given in parentheses identify the corresponding frames in Videos 1 – 6 (available at
JCB • VOLUME 184 • NUMBER 5 • 2009 642
CycB protein ( Stif er et al., 1999 ). Wild-type (2 × CycB) and 1 ×
CycB embryos were injected in one pole at approximately cycle
9 with CycA and CycB3 dsRNA and followed in real time. The
regions in which dsRNA was injected will be referred to as 2 ×
CycB only and 1 × CycB only. In the 2 × CycB – only case ( Fig. 2 A ),
the nuclei go through mitosis, whereas in our previous work, we
showed that knockdown of all three mitotic cyclins arrested nu-
clei in interphase ( McCleland and O ’ Farrell, 2008 ). The inter-
mediate 1 × CycB – only region displayed a new phenotype not
We quanti ed timing through cycle 12. Interphase of 1 ×
CycB – only regions was essentially the same length as that of
2 × CycB – only embryos ( Fig. 3 A ), after which the nuclei began
to condense into mitotic chromosomes, a hallmark of mitotic
entry ( Fig. 3 and Video 9, available at http://www.jcb.org/cgi/
content/full/jcb.200810012/DC1). However, rather than progress-
ing through mitosis, after formation of prophase chromosomes
( Fig. 3 C , 08:01), 1 × CycB – only nuclei appeared to decondense
and revert back to interphase ( Fig. 3 C , 12:31). Prophase chro-
matin condensation was only evident for ⵑ 2 min. The failure to
see a signi cant interphase extension upon reduction of CycB
dose and the abortive attempt at mitosis challenge two ideas: (1)
that cyclin accumulation directly times mitotic entry and (2) that
all mitotic events occur at a single threshold of cyclin/Cdk.
Nuclei exhibited an unusual morphology after the aborted
mitosis. The majority of chromatin was localized at the nuclear
periphery ( Fig. 3 D ). Although appearing somewhat condensed,
this chromatin did not stain positive for phosphohistone H3
(Ser10), suggesting that nuclei were not in an extended prophase
(Fig. S2 B, top, available at http://www.jcb.org/cgi/content/
Nuclei remained in this atypical state for approximately
the same time as the previous interphase ( ⵑ 12 min) at which
time mitotic chromosomes reformed, phosphohistone H3 staining
In conclusion, our data suggest specializations in the roles
of cyclins, especially CycA and CycB3, which appear to be par-
ticularly involved in early and late mitotic events, respectively.
Though strikingly evident in the data, such specialization is in-
complete because redundancies imply overlap in many activities
of the cyclins ( Knoblich and Lehner, 1993 ). Clearly, the origins
of cyclin specialization are complex and will not only re ect spe-
ci c differences in cyclin activity ( Parry et al., 2003 ) but also
each cyclin ’ s spatial and temporal function ( Royou et al., 2008 ).
Embryos operating on a single dose
of cyclin enter mitosis punctually but
The accumulation of mitotic cyclins to a threshold has been sug-
gested to directly time Cdk activation and mitotic entry ( Murray
and Kirschner, 1991 ). Cyclins do in uence mitotic timing in early
syncytial Drosophila embryos, as shown previously by alterations
in interphase length after changes in cyclin maternal gene dose
( Edgar et al., 1994 ; Stif er et al., 1999 ; Ji et al., 2004 ; Crest et al.,
2007 ) and by our nding that RNAi knockdown of any two cyclins
prolonged interphase, in the most dramatic case, to 180% of normal
in cycle 13 (after knockdown of CycB and CycB3; unpublished
data). However, the previous dose experiments did not change
interphase in proportion to the change of gene dose as might be ex-
pected by a direct regulation by accumulation of the cognate cyclin.
This lack of parallel might be explained by the ability of all three
cyclins to induce mitosis. Accordingly, previous dose changes
would be expected to give complex results because they would shift
the balance among different cyclins, each with a seemingly differ-
ent ability to induce mitosis. To simplify the situation and thereby
test whether the quantity of cyclin times mitotic entry, we knocked
down two of the cyclins and reduced the dose of the third.
Embryos derived from a mother heterozygous for a CycB
mutation (1 × CycB) carry a correspondingly reduced level of
Figure 2. RNAi of individual and pairwise
combinations of mitotic cyclin disrupt mitotic
progression. (A) Embryos expressing histone-
GFP were injected with the indicated dsRNA
and followed in real time from mitosis of cycle
11 to the beginning of cycle 14. Mean times
for mitosis 13 are presented. The beginning
of prophase was deﬁ ned by the onset of
chromatin condensation, and metaphase was
deﬁ ned by the complete alignment of chro-
mosomes until anaphase onset. The asterisk
indicates absence of metaphase. Error bars
represent SD. (B) Model for coupling mitotic
events to cyclin function and destruction. The
three mitotic cyclins are temporally degraded
within mitosis: CycA before metaphase, CycB
at the metaphase – anaphase transition, and
CycB3 during anaphase. Our cyclin RNAi
experiments suggest that each cyclin has a
specialized function for distinct mitotic steps.
Coordinating cyclin function and destruction
may guide mitotic progression.
643CELL CYCLE EFFECTS AFTER CYCLIN REDUCTION • McCleland et al.
(Fig. S3 B). 1 × CycA embryos injected with CycB and CycB3
RNAi exhibited a slight interphase delay compared with 2 ×
CycA – only embryos but nonetheless completed nuclear divi-
sion (Fig. S3). Because the timing of mitotic entry as judged by
chromosome condensation is relatively unaffected by the change
in gene dose, whereas the success of mitosis is affected, we con-
clude that a rise in cyclin function to a threshold is not the direct
timer of mitotic entry in Drosophila embryos.
Centrosome division accompanies the
abortive mitosis supported by a single
maternal dose of CycB
To examine centrosome behavior in conjunction with the abor-
tive mitosis, we constructed ies with a single dose of maternal
CycB that expressed either GFP-Polo or transforming acidic
coiled-coil (TACC) – GFP to mark centrosomes. In 1 × CycB –
only embryos, GFP-Polo moved into the nucleus and faintly
decorated kinetochores during the aborted mitosis in cycle 12
was evident, and nuclei progressed through mitosis ( Fig. 3 ; and
Fig. S2 B, bottom). When the 1 × CycB – only region nally pro-
gressed through mitosis, timing defects in mitosis were very
similar to those observed in 2 × CycB – only regions ( Fig. 3 A )
except that chromosomes from neighboring nuclei frequently
collided during anaphase ( Fig. 3 C and Video 9). Our interpreta-
tion of this successful second try at mitosis is that after two cell
cycle intervals, a single dose of cyclin is able to provide a level
of cyclin function achieved by two doses in one cell cycle inter-
val. Accordingly, the second mitosis is much like that seen in 2 ×
CycB – only regions except for the colliding anaphases, which
we attribute to the centrosome division that accompanies the
abortive mitosis (see Centrosome division accompanies … ).
Using a similar strategy, we examined 1 × CycA and 1 ×
CycB3 embryos (Fig. S3, available at http://www.jcb.org/cgi/
content/full/jcb.200810012/DC1). 1 × CycB3 embryos, when
injected with CycA and CycB RNAi, exhibited a curtailed nu-
clear division similar to that described in 1 × CycB – only regions
Figure 3. Embryos relying on one maternal dose of CycB exhibit timely but abortive mitosis. (A) Embryos expressing histone-GFP and containing either
one or two maternal doses of CycB were injected with CycA and CycB3 dsRNA (represented as 1 × CycB or 2 × CycB, respectively). Mean cell cycle times
during cycle 12 are presented. Embryos injected with control LacI dsRNA are presented for reference. (B) Behavior of mitosis 12 in different regions of a
1 × CycB embryo after CycA and CycB3 dsRNA injection at one pole (left). (C) Embryos as in B were imaged at a higher resolution during cycle 12 near
the injection site. (D) Single z sections from C during the atypical interphase (12:31).
JCB • VOLUME 184 • NUMBER 5 • 2009 644
kinetochores were paired, suggesting that sister chromatid co-
hesion was not removed during the aborted mitosis 12 ( Fig. 4 A ).
In this report, we have examined the cell cycle conse-
quences of progressive reductions in mitotic cyclin during the
Drosophila blastoderm divisions. When we nominally reduced
cyclin function to the contribution of a single maternal gene,
embryos attempted mitosis without triggering or completing all
mitotic events. Therefore, there is not a common threshold of
cyclin function for all mitotic events. Moreover, because mito-
sis is attempted at about the normal time despite cyclin knock-
down, the accumulation of cyclin does not appear to be the
direct timer of mitosis. It remains unclear what times interphase
in the Drosophila blastoderm cycles, but inputs from S phase
and/or centrosome duplication might well have important roles
( Sibon et al., 1997 ; Ji et al., 2004 ; Crest et al., 2007 ; McCleland
and O ’ Farrell, 2008 ).
Materials and methods
Drosophila strains were grown as described previously ( McCleland and
O ’ Farrell, 2008 ). Flies expressing histone H2AvD-GFP, GFP-Polo (Flytrap no.
CC01326), and TACC-GFP were used for live embryo analysis ( Clarkson
just as GFP-Polo does at the start of normal mitosis ( Fig. 4 A ,
04:25; and Video 10, left). GFP-Polo subsequently retreated
from the nucleus without evident kinetochore separation ( Fig. 4 A ,
14:54). Centrosomes divided following retreat of GFP-Polo
from the nucleus ⵑ 8 min after the initial GFP-Polo nuclear
in ux ( Fig. 4 A , 14:54; and Video 10, left). Some sister centro-
somes separated only 2 μ m, whereas others separated and moved
around the nucleus. Embryos expressing TACC-GFP corrobo-
rated these results and additionally showed the persistent exclusion
of TACC-GFP in the nucleus, suggesting that nuclear envelope
breakdown did not occur ( Fig. 4 B ; and Video 10, right, available
Thus, 1 × CycB – only regions exhibit prophase chromatin con-
densation, GFP-Polo movements, and centrosome division with-
out reaching a cyclin threshold for nuclear division.
When nuclei reentered mitosis after the initial aborted at-
tempt, they did so with an extra pair of centrosomes and often had
multipolar spindles ( Fig. 4 A , 18:24 and 22:55). Each of the four
centrosomes split again in late anaphase, indicating that centrioles
had replicated during the atypical interphase (Video 10). In con-
trast, based on the number of kinetochores, it did not appear that
chromosomes had replicated again. Moreover, GFP-Polo – labeled
Figure 4. Centrosome division occurs in concert with the abortive attempt at mitosis in 1 × CycB – only embryos. (A) 1 × CycB embryos expressing GFP-Polo
were injected with CycA and CycB3 dsRNA. Dashed circles highlight individual nuclei and associated centrosomes. Arrows point to paired kinetochores
during the second successful mitoses. (B) 1 × CycB embryos expressing TACC-GFP were injected with CycA and CycB3 dsRNA.
645CELL CYCLE EFFECTS AFTER CYCLIN REDUCTION • McCleland et al.
Submitted: 2 October 2008
Accepted: 4 February 2009
Barros , T.P. , K. Kinoshita , A.A. Hyman , and J.W. Raff . 2005 . Aurora A activates
D-TACC – Msps complexes exclusively at centrosomes to stabilize centro-
somal microtubules. J. Cell Biol. 170 : 1039 – 1046 .
Buszczak , M. , S. Paterno , D. Lighthouse , J. Bachman , J. Planck , S. Owen , A.D.
Skora , T.G. Nystul , B. Ohlstein , A. Allen , et al . 2007 . The carnegie protein
trap library: a versatile tool for Drosophila developmental studies. Genetics .
175 : 1505 – 1531 .
Clarkson , M. , and R. Saint . 1999 . A His2AvDGFP fusion gene complements a lethal
His2AvD mutant allele and provides an in vivo marker for Drosophila chro-
mosome behavior. DNA Cell Biol. 18 : 457 – 462 .
Crest , J. , N. Oxnard , J.Y. Ji , and G. Schubiger . 2007 . Onset of the DNA replication
checkpoint in the early Drosophila embryo. Genetics . 175 : 567 – 584 .
Echard , A. , and P.H. O ’ Farrell . 2003 . The degradation of two mitotic cyclins contrib-
utes to the timing of cytokinesis. Curr. Biol. 13 : 373 – 383 .
Edgar , B.A. , and P.H. O ’ Farrell . 1989 . Genetic control of cell division patterns in the
Drosophila embryo. Cell . 57 : 177 – 187 .
Edgar , B.A. , and P.H. O ’ Farrell . 1990 . The three postblastoderm cell cycles of
Drosophila embryogenesis are regulated in G2 by string. Cell . 62 : 469 – 480 .
Edgar , B.A. , F. Sprenger , R.J. Duronio , P. Leopold , and P.H. O ’ Farrell . 1994 .
Distinct molecular mechanism regulate cell cycle timing at successive stages
of Drosophila embryogenesis. Genes Dev. 8 : 440 – 452 .
Evans , T. , E.T. Rosenthal , J. Youngblom , D. Distel , and T. Hunt . 1983 . Cyclin: a
protein speci ed by maternal mRNA in sea urchin eggs that is destroyed at
each cleavage division. Cell . 33 : 389 – 396 .
Ferrell , J.E. Jr ., M. Wu , J.C. Gerhart , and G.S. Martin . 1991 . Cell cycle tyrosine
phosphorylation of p34cdc2 and a microtubule-associated protein kinase
homolog in Xenopus oocytes and eggs. Mol. Cell. Biol. 11 : 1965 – 1971 .
Hartley , R.S. , R.E. Rempel , and J.L. Maller . 1996 . In vivo regulation of the early
embryonic cell cycle in Xenopus . Dev. Biol. 173 : 408 – 419 .
Holloway , S.L. , M. Glotzer , R.W. King , and A.W. Murray . 1993 . Anaphase is initi-
ated by proteolysis rather than by the inactivation of maturation-promoting
factor. Cell . 73 : 1393 – 1402 .
Jacobs , H.W. , J.A. Knoblich , and C.F. Lehner . 1998 . Drosophila cyclin B3 is re-
quired for female fertility and is dispensable for mitosis like cyclin B. Genes
Dev. 12 : 3741 – 3751 .
Ji , J.Y. , J.M. Squirrell , and G. Schubiger . 2004 . Both cyclin B levels and DNA-
replication checkpoint control the early embryonic mitoses in Drosophila .
Development . 131 : 401 – 411 .
Knoblich , J.A. , and C.F. Lehner . 1993 . Synergistic action of Drosophila cyclins A
and B during the G2-M transition. EMBO J. 12 : 65 – 74 .
Lehner , C.F. , and P.H. O ’ Farrell . 1989 . Expression and function of Drosophila
cyclin A during embryonic cell cycle progression. Cell . 56 : 957 – 968 .
Lehner , C.F. , and P.H. O ’ Farrell . 1990 . The roles of Drosophila cyclins A and B in
mitotic control. Cell . 61 : 535 – 547 .
McCleland , M.L. , and P.H. O ’ Farrell . 2008 . RNAi of mitotic cyclins in
Drosophila uncouples the nuclear and centrosome cycle. Curr. Biol.
18 : 245 – 254 .
Moutinho-Santos , T. , P. Sampaio , I. Amorim , M. Costa , and C.E. Sunkel . 1999 .
In vivo localisation of the mitotic POLO kinase shows a highly dynamic
association with the mitotic apparatus during early embryogenesis in
Drosophila . Biol. Cell . 91 : 585 – 596 .
Murray , A.W. 2004 . Recycling the cell cycle: cyclins revisited. Cell . 116 : 221 – 234 .
Murray , A.W. , and M.W. Kirschner . 1989 . Cyclin synthesis drives the early em-
bryonic cell cycle. Nature . 339 : 275 – 280 .
Murray , A.W. , and M.W. Kirschner . 1991 . What controls the cell cycle? Sci. Am.
264 : 56 – 63 .
O ’ Farrell , P.H. 2001 . Triggering the all-or-nothing switch into mitosis. Trends
Cell Biol. 11 : 512 – 519 .
O ’ Farrell , P.H. , B.A. Edgar , D. Lakich , and C.F. Lehner . 1989 . Directing cell di-
vision during development. Science . 246 : 635 – 640 .
Parry , D.H. , and P.H. O ’ Farrell . 2001 . The schedule of destruction of three mi-
totic cyclins can dictate the timing of events during exit from mitosis.
Curr. Biol. 11 : 671 – 683 .
Parry , D.H. , G.R. Hickson , and P.H. O ’ Farrell . 2003 . Cyclin B destruction trig-
gers changes in kinetochore behavior essential for successful anaphase.
Curr. Biol. 13 : 647 – 653 .
Reber , A. , C.F. Lehner , and H.W. Jacobs . 2006 . Terminal mitoses require nega-
tive regulation of Fzr/Cdh1 by cyclin A, preventing premature degrada-
tion of mitotic cyclins and string/Cdc25. Development . 133 : 3201 – 3211 .
and Saint, 1999
; Barros et al., 2005 ; Buszczak et al., 2007 ). Mitotic cyclin
gene mutants CycA
, and CycB3
were used in experiments in
which maternal gene dose was reduced to a single copy ( Knoblich and Lehner,
1993 ; Jacobs et al., 1998 ). Each mutant was crossed to ﬂ ies containing
H2AvD-GFP to generate the following stocks: (a) w ; ; CycA
GFP /TM6, Tb , (b) w ; CycB
, H2AvD-GFP / Cyo , and (c) w ; ; CycB3
Ser , H2AvD-GFP . To facilitate these crosses, the original p-element contain-
ing H2AvD-GFP ( Clarkson and Saint, 1999 ) was mobilized from the third
chromosome onto other chromosomes by crossing these ﬂ ies to ﬂ ies express-
ing the ⌬ 2-3 transposase. CycB
mutants were crossed to ﬂ ies expressing
GFP-Polo and TACC-GFP to generate the following stocks: (a) w ; CycB
Cyo ; GFP-Polo /TM6, Ser , Tb and (b) w , TACC-GFP ; CycB
/ Cyo .
Embryo manipulation and dsRNA production
Embryos were collected and processed for injection as described previ-
ously ( McCleland and O ’ Farrell, 2008 ). dsRNA was generated as de-
scribed previously ( McCleland and O ’ Farrell, 2008 ).
Live analysis of cell cycle progression in embryos
In most experiments shown, embryos were injected at one pole. The sever-
ity of phenotypes increased with time after injection. For example, after
RNAi of CycA and CycB (Video 3), the ﬁ rst mitosis showed a shortened
metaphase, whereas in the next mitosis (cycle 13), there was no obvious
metaphase. Additionally, phenotypes were most severe near the point of
injection. In some cases, marked differences occurred in the behavior of
nuclei at the interface between affected and unaffected territories. For ex-
ample, the interface nuclei between 1 × CycB – only regions and the regions
unaffected by the RNAi did not reenter mitosis ( Fig. 3 B and Fig. S2 A). We
imagine that the neighboring nuclei in the uninjected region inﬂ uence cell
cycle behavior at the edges of the 1 × CycB – only region, perhaps by lo-
cally promoting cyclin destruction in conjunction with their mitotic program.
In other experiments, we controlled for such neighbor effects by depositing
dsRNA along the length of the embryos. These experiments gave a uniform
phenotype identical to that observed near the point of injection after local
injection. This control indicates that the neighbor effects do not extend long
distances and do not contribute to the phenotypes described.
Embryos were imaged on an inverted microscope (DM 1RB; Leica)
equipped with a spinning disk confocal unit (CSU10; Yokagowa), 100 ×
Plan Fluotar 1.3 NA and 40 × Plan Fluotar 0.7 NA objectives (Leica), a
camera (Orca AG; Hamamatsu Photonics), and Volocity 4 acquisition soft-
ware (PerkinElmer). All images and videos were processed in Volocity 4
software and unless otherwise noted are presented as extended focused
images. Image capture rates are indicated on each video. Time is dis-
played in hours:minutes:seconds. Most image stacks were collected at
0.5 – 1.5 μ m over a 5 – 10- μ m range using a controlled stage (MS-2000;
Applied Scientiﬁ c Instrumentation).
At least three independent injections were performed for each ex-
periment shown. In each injection/experiment, an x-y stage facilitated the
ﬁ lming of multiple embryos (usually greater than ﬁ ve embryos). We used a
minimum of four embryos in experiments in which cell cycle times were
quantiﬁ ed ( Figs. 2, 3 , and S3). Usually, times were also collected from the
uninjected end of the embryo and used for an internal reference.
Fixed analysis and immunoﬂ uorescence
Embryos injected with dsRNA were incubated for the appropriate time,
ﬁ xed in a mixture of 37% formaldehyde and heptane for 10 min, and
transferred to methanol before embryo hand devitellinization. Embryos
were rehydrated in phosphate-buffered saline with 0.1% Triton X-100 and
processed for immunoﬂ uorescence. Embryos were labeled with phospho-
histone H3 (Ser10) antibody (Millipore) and Alexa Fluor 546 – conjugated
goat anti – rabbit antibody (Invitrogen). DNA was labeled with Hoescht
Online supplemental material
Fig. S1 illustrates mitotic defects upon knockdown of CycB. Figs. S2 and S3
provide further characterization of embryos operating on one dose of mi-
totic cyclin. Videos 1 – 8 show real-time videos of embryos in Figs. 1 and S1
progressing through mitosis after cyclin knockdown. Videos 9 and 10 are
real-time videos of embryos in Figs. 3 and 4 in which nuclei attempt mitosis
with a single maternal dose of CycB. Online supplemental material is avail-
able at http://www.jcb.org/cgi/content/full/jcb.200810012/DC1.
We thank the O ’
Farrell laboratory (especially Tony Shermoen and Soo-Jung Lee)
This work was supported by a National Institutes of Health training grant
(GM078710) to M.L. McCleland and a National Institutes of Health grant
(GM037193) to P.H. O ’ Farrell.
JCB • VOLUME 184 • NUMBER 5 • 2009 646
Royou , A. , D. McCusker , D.R. Kellogg , and W. Sullivan . 2008 . Grapes(Chk1)
prevents nuclear CDK1 activation by delaying cyclin B nuclear accumu-
lation. J. Cell Biol. 183 : 63 – 75 .
Russell , P. , and P. Nurse . 1986 . cdc25+ functions as an inducer in the mitotic
control of ssion yeast. Cell . 45 : 145 – 153 .
Sibon , O.C. , V.A. Stevenson , and W.E. Theurkauf . 1997 . DNA-replication check-
point control at the Drosophila midblastula transition. Nature . 388 : 93 – 97 .
Sigrist , S. , H. Jacobs , R. Stratmann , and C.F. Lehner . 1995 . Exit from mitosis is
regulated by Drosophila z z y and the sequential destruction of cyclins A,
B and B3. EMBO J. 14 : 4827 – 4838 .
Stif er , L.A. , J.Y. Ji , S. Trautmann , C. Trusty , and G. Schubiger . 1999 . Cyclin A and
B functions in the early Drosophila embryo. Development . 126 : 5505 – 5513 .
Su , T.T. , F. Sprenger , P.J. DiGregorio , S.D. Campbell , and P.H. O ’ Farrell . 1998 .
Exit from mitosis in Drosophila syncytial embryos requires proteolysis
and cyclin degradation, and is associated with localized dephosphoryla-
tion. Genes Dev. 12 : 1495 – 1503 .
Yang , D. , H. Lu , and J.W. Erickson . 2000 . Evidence that processed small dsRNAs
may mediate sequence-speci c mRNA degradation during RNAi in
Drosophila embryos. Curr. Biol. 10 : 1191 – 1200 .